Optimization of post methanated distillery effluent (PMDE...

Optimization of post methanated distillery Optimization of post methanated distillery Optimization of post methanated distillery Optimization of post methanated distillery effluent (PMDE effluent (PMDE effluent (PMDE effluent (PMDE ) ) ) ) in biphasic treatment system in biphasic treatment system in biphasic treatment system in biphasic treatment system

of bacteria and wetland plants for of bacteria and wetland plants for of bacteria and wetland plants for of bacteria and wetland plants for environmental safety environmental safety environmental safety environmental safety

Chapter 7

Biphasic treatment of PMDE…

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Optimization of post methanated distillery effluent (PMDE) in biphasic treatment system of bacteria and wetland plants for

environmental safety

(A) Optimization of PMDE degradation and decolourisation in biphasic treatment of bacteria and mixed population of wetland plant in design treatment system Complex industrial wastewater i.e. distillery wastewater that are treated by biological means are good candidate to be addressed by constructed wetland treatment system. Wetlands are considered sink for contaminates due to its remediation potential for wide varieties of pollutants. Wetland plants which accumulate different environmental pollutants included heavy metals either through phytostabilization or phytoextraction process. The proper functioning of the wetland system is dependent on the interactions between plants, soil, wastewater characteristics, microorganisms and operational conditions (Aguilar et al., 2008). Several designed features may affect the processes occurring in a constructed wetland (Kadlec et al., 2000); for instance, changes in design and loading regime may improve the oxidation in constructed wetland system. It has been suggested that macrophyte species also affect the pollutant removal efficiency in constructed wetland system. Recently, the application of constructed wetland treatment has been reported promising for the industrial wastewater remediation/treatment (Chen et al., 2006). Plant roots and rhizomes are important for the microbial transformation processes of wastewater purification (Stottmeister et al., 2003). Microbial assemblages can be found as a biofilm on substrate and root surfaces. Many parameters affect biofilm structure, especially nutrient availability or other environmental conditions (Kierek-Pearson and Karatan, 2005). Use of constructed wetland treatment process is accepted as eco-friendly, cost-effective and energetically sustainable technique compared with the conventional wastewater treatment system. Constructed wetland treatment system generally fall in to two categories: (a) horizontal flow system (HFS) and (b) vertical flow system (VFS). HFS may be free water surface system or subsurface system depending up on their designing. Free water surface system can mimicked the pollutants by flow over the bed surface and it filter through a dense stand of aquatic plants, while in subsurface flow water system consisting of an excavated, but usually lined shallow basin containing pebbles, gravels and uppermost layer is sand (EPA, 1998). Lower most zone containing pebbles gives ample opportunity for growing network of fibrous root, where rhizospheric bacteria makes biofilm and it act as biofilter. Thus, during the treatment of wastewater, water flows under subsurface of designed layer. In contrast, VFS, wastewater during treatment moves either down flow or up flow. But, it required comparatively higher operational maintenance cost compared to HFS due to the necessity to pump wastewater intermittently on the wetland surface containing plants. The wastewater is fed into large batches and then it percolates down through sand medium. The new batch is fed only after all the water percolates and the bed is free of water. This enables diffusion of oxygen from the air into the bed. As a result, VFS are for more


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aerobic than HFS wetland system and provide suitable condition for nitrification, while HFS provide more suitable condition for denitrification due to semi-anaerobic condition. VFS is very effective in removing organic and suspended solids. Moreover, it required more land area. Therefore, VFS are very often using to treat domestic and municipal wastewater or wastewater in which discharge limit of ammonia-nitrogen is fixed. Hence, in this study constructed system has been designed with subsurface horizontal flow wastewater treatment system, which can degrade variety of pollutants in semi-aerobic condition due to specific microbial biofilm on the root zone of wetland plants.

Since, PMDE containing different types of pollutants which do not easily decolorized by single step treatment. Therefore, biphasic treatment of complex wastewater using bacteria and constructed wetland might be more promising. Hence, bacterial pre-treated effluent was integrated with wetland treatment system consisting different group of potential wetland plants (Phragmites cummunis, Typha anguistifolia and Cyperus esculentus). The commonly growing wetland plants in industrial contaminated wetland has been selected as described in previous chapter six and acclimatized in constructed wetland system for the improvement of decolourisation and degradation of PMDE in integrated bacteria and wetland treatment system. T. anguistifolia and C. esculentus is faster growing plants than the P. cummunis. However, P. cummunis has double advantage; first it has bigger vegetable growth and second has strong root system. Moreover, advantage of mixed wetland plants has differential growth and tolerance potential. This increases the tolerance of organic and hydraulic load. Furthermore, structural and physiological variation will offer wide opportunity for different group of micro-organism in their rhizospheric zone. This will established more effective biofilter system for removal of recalcitrant compounds present in PMDE.

A.7.1. Materials and methods

A.7.1.1. Construction of the wetland treatment system and acclimatization of the plants

The wetland treatment system was constructed in an 8 x 21 m area located at the Indian Institute of Toxicology Research, India, where in 19 x 4 m area, a zig-zag channel was constructed using cement to prevent the seepage. Upstream channel depth and width were kept at 50 and 40 cm, respectively, while the downstream dept and width were kept at 58 and 40 cm, respectively, thereby maintaining a 0.88% slope that promoted hydraulic flow. The horizontal bed was filled with pebbles, coarse gravel and sand particles, each layer being 8 cm deep, as in the design of Swindell and Jackson (1990) and the EPA design manual (EPA, 1998) as shown below. Naturally growing wetland plants (P. cummunis, T. angustifolia and C. esculentus) were collected from a wetland area polluted by PMDE. The young buds (plantlets) of the subterranean rhizomes of this species were acclimatized for more than 60 days in a constructed free water surface flow wetland system. P. cummunis, T. angustifolia and C. esculentus occupied the region E1, E2 and E3, respectively in constructed wetland treatment system. During this period, the plants were fed with 5% (v/v) Hoagland’s solution (Hoagland and Arnon, 1938) for 30 days; the solution contained (in g/l) 1.18 Ca(NO3)2

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0.51 KNO3, 0.14 K2HPO4, 0.49 MgSO4.7H2O, 0.005 FeCuH4O6, 0.0029 H3BO3, 0.0018

MnCl2.4H2O, 0.0022 ZnSO4, 0.00008 CuSO4

.5H2O and 0.00002 H2MoO4. Subsequently, the plants were further acclimatized for 30 days by means of surface flow circulation containing PMDE mixed with the above mentioned Hoagland’s solution (30:70 v/v).

Fig. 7.1. Schematic diagram showing biphasic treatment of PMDE for environmental safety

The diluted effluent was re-circulated using a pump to maintain natural wetland conditions. The upstream flow rate was 2 L/min versus 1.5 L/min downstream. To ensure stable performance of the study system, three wetland systems with subsurface flow were installed for each of four tested effluent concentrations (10%, 30%, 50% and 100%). Schematic diagram showing biphasic treatment of PMDE is shown in Fig. 7.1.

A.7.1.2. Experimental protocol

A.7.1.2.1. Inoculums preparation and bacterial decolourisation of PMDE in designed reactor

Bacterial consortium growing in fermenter having 7.5 L capacity was used as inoculums for PMDE decolourisation at pilot scale. For seed preparation of bacterial consortium, 8% of developed bacterium [Proteus mirabilis (4) : Bacillus sp.(3) : Raoultella planticola (1) : Enterobacter sakazakii(1)] was inoculated in sterilized 5.0 L modified GPYM medium containing glucose (1%), peptone (0.1%), yeast extract (0.1), K2HPO4 (0.1%) and MgSO4

.7H2O (0.05%) amended with PMDE (20%) and incubated at 35 ± 2 ºC and 180 rpm in

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Collecting tank after decolorisati-on

A : Bioreactor for bacterial treatment of distillery effluent at optimized condition B : Constructed wetland consisting with mixed vegetation C : Collecting tank with decolorized distillery effluent D : Step for environmental safety assessment and scope for decolorized effluent recycling E1 : Phragmites cummunis zone E2 : Typha anguistifolia zone E3 : Cyperus esculentus zone

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fermentor (7.5 L). The original PMDE was considered to represent a concentration of 100%. Three additional concentrations of PMDE were created (10%, 30% and 50% v/v) by dilution with tap water and modified GPYM medium was added to provide the minimal salt levels required to support bacterial growth. Bacterial inoculum (8% v/v) was obtained from fermenter in the log growth phase (1.1 x 107 cells/ml) and the bacteria were inoculated into 5000 L bioreactors containing 3000 L of effluent at the desired concentration (10%, 30%, 50% and 100%). One reactor was without bacterial inoculums served as the control. The cultures were incubated at 180 rpm for 5 consecutive days with continuous aeration. After 5 days, aeration was stopped and the reactors were left overnight to permit settling of the bacterial biomass. Supernatant of this treatment was designated as BT. Two parallel sets of bioreactors were operated for the control and experimental treatments to maintain continuous flow with and without bacterial pretreatment of the PMDE in the constructed wetland systems.

A.7.1.2.2. Integration of bacterial pretreated effluent with constructed wetland plant treatment system

The supernatant from the bacterial pretreatment of the effluent, at all four concentrations (10%, 30%, 50% and 100% v/v), was fed into a constructed subsurface flow wetland system, as described in section A.7.1.1 with an upstream flow rate of 2 L/min and a downstream flow rate of 1.5 L/min. The bacteria-treated effluent was circulated for up to 7 days until the BOD of the effluent reached a value of 100 mg/l (the permissible limit for terrestrial disposal; EPA, 1986). The effluent discharged after bacterial and wetland plant treatment was designated as WT. The physico-chemical parameters of the wetland water (BOD, COD, phosphate, nitrogen and colour) were periodically analyzed to assess the performance of the wetland plants. For each effluent concentration, another set of effluent with the same concentration but without bacterial pretreatment was also circulated through a separate wetland system to serve as a control.

A.7.1.3. Optimization of flow rate (FR) and hydraulic retention time (HRT) for PMDE decolourisation in constructed wetland treatment system

The proper functioning of wetland treatment system depends on the interaction between the plants, microbes (rhizospheric zone), wastewater and operational condition. The flow rate and hydraulic retention time (HRT) determine the interaction of wastewater with plant and microbes. In our study decolourisation was optimized at different flow rate from 10-30 m3/d by putting the controlling knob at connecting points of bioreactor (bacterial treatment) and upstream part of constructed wetland system.

A.7.1.4. Physico-chemical analysis of the effluent before and after treatment

All the physico-chemical parameters before and after PMDE treatment was periodically monitored by analyzing pH, COD, BOD and colour as per standard methods described in APHA, 2005. Nitrogen as nitrate, phosphate, sodium and chloride were measured


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through Orion meter as per operating manual. During the physico-chemical analysis the sulphate and heavy metals were also measured in pre and post treated sample prior to its final discharge. HPLC analysis of different stages of PMDE was also analyzed as described in chapter 3.

A.7.1.5. Effect of seasonal variation on PMDE decolourisation and degradation in designed biphasic treatment system

Since the temperature is an important parameters for the growth of plant and microbes. Therefore, the performance evaluation of treatment process was also done in different season due to temperature variation. The PMDE decolourisation process was monitored and assessed in designed treatment system in summer (April-June), monsoon (July-September) and winter (November to January).

A.7.2. Results and discussion

A.7.2.1. Bacterial treatment of PMDE (BT)

Bacterial consortium comprising Proteus mirabilis (4) : Bacillus sp.(3) : Raoultella planticola (1) : Enterobacter sakazakii (1) decolourized 30% PMDE up to 60% at optimized condition of nutrient and different parameters after 4 days of incubation. Optimized nutrient was 1% glucose, 0.1% peptone, 0.1% yeast extract, 0.1% K2HPO4, 0.05% MgSO4

.7H2O and 180 rpm was optimum shaking speed. Bacterial consortium was unable to decolorized PMDE higher than 50%. This might be due to higher concentration of toxic compounds which inhibit the bacterial growth. During bacterial treatment along with colour other pollution parameter i.e. COD, BOD and TS were also reduced after 4 days incubation (Fig. 7.2 and Table 7.1).

Fig. 7.2. Periodic reduction of colour, COD, ammonia and nitrate after bacterial treatment (BT) in 5000 L bioreactor.

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A.7.2.2. Optimization of different process parameters in constructed wetland treatment system

Supernatant of bacterial treated PMDE was integrated in to constructed wetland treatment system with different flow rate and retention time to obtain the more decolourisation (Fig. 7.3). Flow rate from 10 to 28.8 m3/d showed very slight difference in PMDE decolourisation. However, flow rate at 30 m3/d showed non-significant decolourisation as compared to 28.8 m3/d flow rate (Fig.7.3). In contrast, PMDE decolourisation was greatly affected by HRT. Gradual increases in HRT increases the decolourisation process (Fig. 7.3). The optimum decolourisation was found at 7 days HRT. Beyond this HRT (7 days) decolourisation was constant.

Fig. 7.3. Represents percent color reduction of PMDE at different flow rate (a), hydraulic retention time (b).

A.7.2.3. Physico-chemical properties of different stages of PMDE

Physico-chemical properties of PMDE before and after biological treatment are shown in Table 7.1. PMDE had very high pH (8.53), COD (58,018 mg/l) and BOD (29,120 mg/l). However, in BT these parameters were comparatively decreased, while in WT the foresaid parameters were reduced within permissible limit (Table 7.1). Similarly, the different metals contents were found significantly high in PMDE as compared to BT and WT. This reduction might be due to bacterial treatment bio-transformed the pollutants present in PMDE. It was also interesting to note that bacterial pretreatment followed by phytoremediation with P. cummunis, T. angustata and C. esculentus for 7 days also improved the physicochemical properties of the effluent: the reductions in colour, BOD and COD were 97%, 98.23% and 99% respectively, compared with the control (Fig. 7.4). Suspended solids are retained predominantly by filtration and sedimentation and the removal efficiency was found very high. Different stages of PMDE treatment are shown in Fig. 7.5. BOD, COD, color and nitrogen concentrations all decreased greatly (by 82.39–99%) compared with the control,

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whereas little phosphate reduction was observed. This may have resulted from the low level of dissolved oxygen that is present in the effluent and from partial phosphate solubilization by the rhizospheric bacteria of the wetland plants (Chen et al., 2006). The major removal mechanism for nitrogen in constructed wetland system is denitrification. Removal of ammonia is limited due to lack of oxygen in the filtration bed as a consequence of permanent waterlogged conditions. Phosphorus is removed primarily by ligand exchange reactions, where phosphate displaces water or hydroxyls from the surface of iron and aluminum hydrous oxides. The most important roles of plants in constructed wetland treatment system are provision of substrate (roots and rhizomes) for the growth of attached bacteria, radial oxygen loss (oxygen diffusion from roots to the rhizosphere), nutrient uptake and insulation of the bed surface in cold and temperate regions. The overall results suggested that biotransformation of recalcitrant compounds with organic compounds into such forms that are bioavailable to wetland plants. In addition, studies of cross-flows through the rhizosphere of T. angustata suggest that the plant’s roots acted as a biofilter that immobilized the bacterial biomass (Whiting et al., 2001).

Fig. 7.4. Reduction in pollution levels as a result of bacterial treatment combined with phytoremediation by T. anguistata.

A.7.2.4. Assessment of PMDE decolourisation by HPLC analysis

The HPLC analysis of PMDE, BT and WT samples after different time treatment are shown in Fig. 7.6. Results have shown gradual reduction in peak areas at different time compared to PMDE indicating the degradation and decolourization of PMDE by bacterial consortium and wetland plants (Fig. 7.6).

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Fig. 7.5. Represents to a view of PMDE at industrial scale after extended aeration (a), PMDE decolorization after bacterial treatment in 3000 L capacity reactor (b), view of constructed wetland treatment system after integration of bacterial pretreated PMDE for decolorization(c) and the view of final decolorization PMDE after bacterial and wetland plant treatment in 5000 L capacity reservoir. Table 7.1. Physico-chemical properties of different stages of PMDE

All values represent means (n=5)±SD and in mg/l except EC (dS/m), color (Co-Pt) ; Statistical significance of the PMDE and treated group were evaluated in column by means of ANOVA. Significance levels: a = p < 0.001; b = p < 0.01; c = p < 0.05; ns = p > 0.05. *Parentheses values are permissible limit of different parameters for safe disposal of effluent.

The reduction in peak areas might be largely attributed to the bacterial degradation of complex organic and inorganic pollutants present in PMDE and their subsequent utilization

Parameters Control Bacterial treated

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pH 8.00±0.32 6.50±0.33* 7.00±0.21* 12.51 Colour 60000±3450 35940±806* 1800±465* 97.00 BOD 12,000±360 7074±124* 240±167* 98.23 DO 5.00±0.23 1.90±0.06* 8.50±0.32* -70.00 COD 40,000±387 17500±431* 400±311* 99.00 TSS 28810±365 22075±345* 864±167* 97.00 TDS 16542±264 11023±278* 661±168* 96.00 TS 45352±673 33098±649* 453±333* 99.00 TOC 7.16±0.35 5.54±0.86* 0.85±0.24* 88.00 Sulphate 2654±65.87 1834±68.45* 796±36,85* 70.00 Phosphate 1218±35.57 987±21.73* 487±35.76* 60.00 Potassium 410±18.78 97.56±2.11* 7.40±1.11* 98.00 Sodium 54.12±1.34 32.12±0.61* 27.06±0.95* 50.00 Copper 1.29±0.04 0.58±0.007* 0.10±0.006* 92.00 Cadmium 0.12±003 0.05±0.00* 0.001±0.00* 99.00 Zinc 11.24±0.56 6.60±0.06* 0.67±0.01* 94.00 Iron 50.64±2.53 24.30±0.94* 10.12±0.43* 80.00 Nickel 0.12±0.003 0.08±0.004* 0.03±0.003* 75.00 Manganese 2.94±0.14 1.85±0.23* 0.58±0.03* 80.00

b c d a


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by bacteria and wetland plant rhizosphere microbes as well as wetland plant roots as source of carbon, nitrogen and energy.

Fig. 7.6. Represents to reduction of coloring peak in HPLC analysis at different time after bacterial treatment. (a), and after wetland plant treatment of bacterial pre-treated PMDE.

A.7.2.5. Seasonal effect on physico-chemical parameter of distillery effluent

Periodic monitoring of BOD, COD and colour at 15 days interval showed constant reduction pattern in summer and monsoon season after treatment with bacteria and wetland plants as shown in Fig. 7.7a-b. While, the reduction pattern of COD, BOD and colour slowed down in extreme winter (December to January) season as shown in Fig. 7.7c. This might be due to favorable temperature for growth of plant and micro-organism in summer and

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monsoon. In contrast, extreme low temperature reduces the metabolic rate of plant and micro-organism. The plant growth accumulates heavy metals and many organic compounds directly from the effluent during treatment. Besides, microbial growth in rhizosphere facilitates reduction of phosphate, nitrogen, organic load by solubulizing their salt, which consequently reduced the colour.

Fig. 7.7 (a-e): Periodic comparison of BOD,COD and colour value with control during the effluent treatment with bacterial followed by constructed wetland plant in summer, monsoon, and winter season. C : values of without wetland plant treated effluent (Control); T : values of effluent treated with bacteria and wetland plants (treated)


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(B) Assessment of bacterial pretreatment for removal of heavy metals during detoxification of post-methanated distillery effluent by Typha angustata L.

During the fermentation process, the fermentable sugar is converted into alcohol, leaving the complex non-degradable organic residues as distillery waste after distillation. Water-soluble hemicelluloses, proteins, gums, non-sugar organic compounds and minerals from the cane juice remain in the spent washwater in their original or converted forms and exert a high chemical oxygen demand. These materials subsequently undergo anaerobic digestion at conventional secondary treatment plants, where phenolics and butyric acid are produced by reducing sugar, with the emission of methane, leaving the sulfides, phenolics and heavy metals. Phenolics and heavy metals inhibit biodegradation of PMDE during tertiary treatment . The heavy metals also form complexes with sulfides, and these complexes contribute to the toxicity of the effluent (Kumar and Chandra, 2004). Therefore, heavy metals must be removed from PMDE to reduce the toxic load created by this waste. Primarily, as a result of difficulties in treating the effluent and the high concentrations of dissolved organic and inorganic matter, a host of schemes for the utilization or treatment of this waste have been proposed (e.g. recycling, direct application to land, evaporation, combustion, aerobic and anaerobic biological treatment, thermal and thermo-chemical methods), but these techniques are either economically impractical or fail to meet the environmental safety standards for the pollutants (Chandra and Pandey, 2001). Recent developments have indicated that hybrid methods that combine partial treatment with two or more steps favour more complete treatment of this effluent (Thompi, 2000). In the present study, we investigated the possibility of using bacterial pretreatment of PMDE to enhance subsequent bioaccumulation of heavy metals by a wetland plant (Typha angustata L.) in a constructed wetland system. Because the bacterial degradation of PMDE should increase the bioavailability of heavy metals and other pollutants as nutrients for wetland plants, this integrated technique offers considerable potential for enhancing the biodegradability of distillery waste and improving the safety of waste disposal.

B.7.1. Experimental setup

Experiment was design for decolourisation of PMDE in two step treatment using another bacterial inoculum in a designed reactor followed by constructed treatment process consisting with T. angustata. Experimental designing was done as described in previous part. However, in this section phytoremediation potential of T. angustata for heavy metal from PMDE was assessed. To determine metal concentrations in the wetland plants, plants were gently uprooted and washed thoroughly with distilled water to remove sand clinging to the roots, followed by rinsing with a 10 mmol/l solution of CaCl2. The plants were then separated into roots, rhizomes and leaves were chopped into small pieces and the resulting biomass was oven-dried at 70 °C for 7 days to constant weight. The dried biomass was then ashed in a muffle furnace at 460 °C for 6 h. The weighed ash from these samples was digested in 2% HNO3 and filtered through a 0.45 m glass fiber filter (AOAC, 2002). One gram of dried and


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sieved sediments was digested with 10 ml of HNO3. If brown fumes longer appeared, 5 ml of HNO3 was added and digestion continued until no brown fumes. The sample was cooled, 2 ml water and 3 ml of H2O2 added and digestion continued followed by EPA method 3050-B (EPA, 1996) and the concentrations of Cd, Cu, Cr, Fe, Mn, Ni, Pb and Zn were measured using an inductively coupled plasma (ICP) spectrophotometer (IRIS Interepid II XDL: Thermo Electron, Waltham, Mass.,USA). The ICP was calibrated before each set of measurements using the procedure specified in the manufacturer’s manual. The heavy metals in the effluent were also analyzed by means of acid digestion, following the standard method for the examination of water and wastewater (APHA, 2005). To analyze the metal contents in bacterial biomass, the bacterial culture suspension was centrifuged at 3000 rpm for 15 min at 4 1C to separate the bacterial biomass as pellets. The bacterial pellets were then washed three times with double-distilled water and dried at 70 °C for 4 days until constant weight. The dry weight of the bacterial biomass was recorded and metal concentrations were determined (following the previously described method for the plant material) and expressed on a dry weight basis. As a control, the heavy metal concentrations of dried bacterial biomass of B. thuringiensis (MTCC 4714) obtained from pure culture was also measured after digestion. To test the data for significance, one-way ANOVA was performed (p<0.05) to compare the control and treated samples at different concentrations of effluent during effluent treatment. Tukey’s test (Ott, 1984) was performed using the Graph Pad software (GraphPad Software, San Diego, Calif.).

B.7.2. Results and discussion

B.7.2.1. Metals in effluent

The PMDE showed high concentrations of heavy metals even after anaerobic digestion (100% concentration). The dominant heavy metal (all concentrations in mg/l) was Fe (84.01) followed by Zn (4.63), Pb (4.45), Cd (2.28), Mn (2.11), Ni (1.24), Cu (0.96) and Cr (0.44). The metal concentrations in PMDE might be released due to corrosion of the metallic vessels at acidic pH during repeated boiling of sugarcane juice and are further concentrated in fermented sugarcane molasses during the production of sugar and alcohol.

B.7.2.2. Biosorption of metals in bacterial biomass

The bacterial biomass showed concentration-dependent accumulation of the metals, with the maximum accumulation at the middle concentrations of effluent (30% and 50%). The bacteria absorbed the most Fe at all tested concentrations, followed by Zn, Cu, Pb, Cd, Cr, Mn and Ni. The maximum bio-sorption of Fe by the bacterial biomass probably resulted from a combination of faster bio-sorption of this metal by the bacteria’s exo-polysaccharides and greater permeability of its cell membrane to Fe, which is an important micronutrient in bacterial metabolic activity (Anita et al., 2004).


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B.7.2.3. Effects of bacterial pretreatment on bioaccumulation of heavy metals by T. angustata

T. angustata was able to grow even at the 100% concentration of effluent pretreated with B. thuriengiensis (MTCC 4714) with no visual toxicity symptoms observed up to 30 days after treatment. In the absence of pretreatment, this concentration produced various visible toxicity symptoms such as reduced growth and rapid yellowing of the leaves. This difference may have resulted from the high load of toxic organo-metallic compounds at this concentration and the ability of bacterial pretreatment to bio-transform the pollutants, resulting in reduced toxicity. The plants grown in 100% effluent without bacterial pretreatment accumulated up to 37.97% of Cd, 34.55% of Cr, 42.09% of Cu, 43.75% of Fe, 41.00% of Mn, 39.73% of Ni, 37.40% of Pb and 43.30% of Zn. The plants grown in various concentrations of bacterial pretreated effluent removed significantly greater (p < 0.01, ANOVA) amounts of all metals. The bioaccumulation of all metals by T. angustata in 100% effluent pretreated with B. thuringiensis reached 71.99% of Cd, 70.45% of Cr, 74.97% of Cu, 76.25% of Fe, 76.99% of Mn, 75.58% of Ni, 70.85% of Pb and 75.25% of Zn. However, the maximum removals of the heavy metals by the biphasic treatment system form 10% and 30% of effluent concentration. Bacterial pretreatment increased the removal of each metal at all tested effluent concentration. The levels of bioaccumulation of various heavy metals by T. angustata across all tested concentrations of effluent after bacterial pretreated were 71.99–99.00% for Cd, 70.45–94.56% for Cr, 74.97–100% for Cu, 76.25–100% for Fe, 76.99–100% for Mn, 75.58–100% for Ni, 70.85–97.07% for Pb and 75.25–100% for Zn. Thus study concluded bacterial pretreatment of PMDE followed by constructed wetland treatment enhanced the phytoremediation of heavy metals from PMDE during decolourisation.

(C) Effect of biologically treated post methanated distillery effluent on seed germination and growth parameters of Vicia faba

Vicia faba has been reported for toxicity assessment in guideline of environmental safety (OECD, 1993). Recent investigations have indicated that land disposal for irrigation of distillery effluent could lead to groundwater contamination and adverse affect crop growth and soil poperties (Jain et al., 2005). Hence, safe land disposals and environmental impact assessment is essential prior to application of any industrial wastewater for ferti-irrigation. Considering the problems in the treatment and disposal of distillery effluent, effluent are polishing prior to land discharge for irrigation in the surrounding area would appear to be more promising. Therefore, PMDE was initially subjected to primary treatment with bacteria. The primary treated PMDE was subjected to constructed wetland treatment system for secondary treatment. Thereafter, in present study the effect of PMDE and biologically treated PMDE on seed germination, physiological parameters, metals translocation and involvement of antioxidants in V. faba were studied.


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C.1. Methodology

C.1.1. Biological treatment of PMDE

PMDE was biologically treated in two steps as described in section 2.2.2. In 1st step PMDE was treated with our previously isolated potential bacterial strains and these samples was designated as (BT). In 2nd stage treatment, sludge was separated after bacterial treatment and supernatant was fed into a constructed subsurface flow wetland system. The bacterial treated PMDE was circulated in constructed surface flow wetland system till the BOD of the effluent reduced up to 100 mg/l (land disposal permissible limits; EPA, 1986). This 2nd step samples were designated as WT.

C.1.2. Effect of different concentration of PMDE, BT and WT on V. faba

The pots (20 X 16 cm) were filled with 7 kg of loamy garden soil. The soaked, uncontaminated and healthy ten seeds were evenly sown in each pot. The pots were irrigated with 50 and 100% concentration of PMDE, BT and WT. This study was replicate in five times along with control (irrigated with tap water).

C.1.3. Physico-chemical analysis of effluent and garden soil

The organic matters (OM), cation exchange capacity (CEC) of treated soil were estimated using method described by Kalra and Maynard (1991). While pH, electrical conductivity (EC) was measured using Orion ion and conductivity meter respectively (Model 960, Model 150) in 1:2.5 w/v suspension (soil: water).

C.1.4. Plant growth and biochemical parameters analysis

Effect of different stages of PMDE (BT and WT) on seed germination was tested as changes in the speed germination; peak value and related growth rate. These were calculated by methods described by Ramana et al. (2002c). Metals content in V. faba was analyzed as per standard methods as described in previous chapter.

C.2. Results and discussion

C.2.1. Effect of PMDE, BT and WT on seed germination of V. faba

In irrigation studies germination was significantly inhibited by 50 and 100% PMDE which was noted 52 and 70% respectively. However, only 30% inhibition was observed in BT, while 50 and 100% WT irrigated V. faba showed 100% germination. This further indicated that biological treatment reduced the pollutants. In contrast, 50 and 100%WT irrigated V. faba showed increased germination speed, peak value and relative growth rate (Table 2). The inhibition of seed germination by PMDE might be due to high osmotic pressure of the effluent which increases with increase effluent concentration. It has been noted, high osmotic pressure of germinating solution inhibit the germination (Ramana et al., 2002c). Metals content after irrigation with biologically treated PMDE was compared to


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control. The data showed that soil accumulated higher concentration of metals in all tested condition after 90 days of irrigation compared with control. But, the 100% PMDE irrigation added higher metals compared with WT irrigated soil. This revealed that biological treatment not only reduced the physico-chemical parameters of PMDE, but also reduced the metals content.

Table 7.2. Effect of different stages of PMDE on seed germination of V. faba

PMDE: post methanated distillery effluent; BT: bacterial treated PMDE; WT: wetland plant treated PMDE.

Continuous irrigation with different concentration of PMDE up to 90 days also increased OM, CEC and EC of soil compared to control. This is because of precipitation of salt in the soil with PMDE application. Hence, the longer indiscriminate application of PMDE may create problem of soil salinity. V. faba irrigated with WT shown significant increase in shoot and root length at all exposure period as compared to control. WT supported better root, shoot, nodule and seed formation in 90 days of V. faba as compared to control (Fig. 7.8). But, the PMDE irrigated V. faba reduced root, shoot length and nodule formation. This may be due to inhibitory effect of different pollutants of PMDE along with metals on plant growth regulators. In addition, in the present study flowering was also severely inhibited in PMDE irrigated V. faba.

Fig. 7.8. Effect of PMDE (50%; 100%), BT (50%; 100%) and WT (50%; 100%) on the growth of V. faba after 90 days irrigation. PMDE: post methanated distillery effluent; BT: bacterial treated PMDE; WT: wetland plant t reated PMDE

Samples Germination percentage

Speed of germination

Peak values Relative growth rate

Control 100.00 7.67 1.23 0.010 50%PMDE 48.32 3.61 0.72 0.008 100%PMDE 30.00 2.66 0.51 0.004 50%BT 80.00 10.03 0.98 0.049 100%BT 70.00 9.32 0.89 0.032 50%WT 100.00 15.53 3.15 0.612 100%WT 100.00 13.78 2.85 0.520

100% WT Control 50% PMDE 50% WT 100% BT 50% BT 100% PMDE


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However, V. faba irrigated with BT showed comparatively better flowering and seed formation as compared to PMDE irrigated V. faba. This revealed that there was also reduction of those pollutants which affect the plant reproduction hormone (flowering and fruiting hormone). The results have shown metals accumulation was increased with increasing concentration of different stages of PMDE and exposure period. The metals content in different parts of V. faba were observed in order root > shoot > leaves at 30 days of exposure. Higher metals accumulation in root might be due to complexation of metals with sulphydrile group, resulted into less translocation of metals to upper parts of plants. However, at 60 and 90 days of exposure the order of accumulation was found as root > leaves > shoot this might be due to metal mobility and transpiration rate. Transpiration rate is major force that drives metal uptake and translocated up to the leaves. The order of metal accumulation was found Fe > Mn > Zn > Ni > Pb > Cu. The metals accumulation was observed least in the seed of WT irrigated V. faba as compared to the BT and PMDE. Seed formation is totally suppress in 100% PMDE irrigated V. faba. The accumulation was found maximum in root part followed by aerial parts, which is in conformity with earlier findings of the other authors (Ebbs and Kochian, 1998). In addition, due to the development of fibrous root system with dense root hairs in terrestrial plants provides ample surface area to absorb large quantities of metals from growth medium (Singh et al., 2004a,b).

Therefore, the present study has shown that continuous irrigation of PMDE leaded to high metals accumulation. This might be probably having lethal effect on plants after a few year of cultivation using PMDE. To overcome these difficulties it is suggested that effluent should be biologically treated which bring down the concentration of phytotoxic substances and this would be also safe for irrigation and wastewater recycling.

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